Serial Electrophoresis

ABSTRACT

Disclosed are methods for performing capillary electrophoresis on two or more nucleic acid samples. The methods employ a forward voltage to move a first sample forward from an inlet to an interrogation region in the capillary, then a backward voltage to move the first sample backward, and then a forward voltage again to move the first sample and a second sample forward. Systems and apparatuses for performing capillary electrophoresis are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefits under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 62/509,618 entitled: SERIALELECTROPHORESIS, filed May 22, 2017, which is herein incorporated byreference in its entirety for all purposes.

BACKGROUND

Capillary electrophoresis systems are used for separation of, e.g., PCRamplicons. In a typical workflow a sample is brought to the one end ofthe capillary where a small portion of the sample is injected as a plug.Typically, high voltage is applied and the charged species in the samplestarts migrating inside the capillary where gel discriminates betweenspecies based on speed; the species migrate such that species withdifferent properties become separated. The polarity of the high voltageis chosen so that species of interest are driven to migrate towards theother end of the capillary where, at some distance before the end, theyare detected. The first species arrives at the location of detection ata particular time, and then the species of interest arrive later,according to their respective properties. The output from the detectorreveals the time of arrival of any of the species in the population,from which information about said species, such as its size, can bediscerned.

In increasing throughput of electrophoresis systems would provide manyscientific and investigative benefits.

SUMMARY

The present disclosure relates to methods, systems, and apparatuses forperforming capillary electrophoresis.

One aspect of the disclosure relates to a method of performing capillaryelectrophoresis of multiple samples. The method includes: introducing afirst sample to an inlet of a capillary containing a separation medium,the first sample including first DNA fragments having a plurality ofdifferent sizes; performing electrophoresis on the first sample in thecapillary, wherein the electrophoresis includes applying a firstsubstantially constant forward polarity electrophoresis voltage to thecapillary while detecting some of the first DNA fragments at aninterrogation region proximate a distal end of the capillary; before allof the first DNA fragments have passed the interrogation region,applying a reverse polarity voltage pulse to the capillary, therebytransporting at least some of the first DNA fragments in the capillarytoward the inlet of the capillary, and thereafter introducing a secondsample to the inlet of a capillary, the second sample including secondDNA fragments having a plurality of different sizes; and applying asecond substantially constant forward polarity electrophoresis voltageto the capillary to simultaneously perform electrophoresis on the secondDNA fragments and the first DNA fragments.

In some implementations, the method further includes introducing aplurality of internal calibrants, along with the first sample, to theinlet of the capillary, wherein each internal calibrant includes alabeled DNA fragment of distinct size. In some implementations, thefirst DNA fragments include amplicons from loci of STRs in a DNA source.

In some implementations, the method further includes amplifying the DNAsource to produce the amplicons from loci of the STRs.

In some implementations, the amplifying is performed on a system thatincludes the capillary.

In some implementations, the system includes a cartridge includingfluidic passages and a thermocycler configured to perform polymerasechain reaction. In some implementations, introducing the second sampleto the inlet of the capillary includes: introducing the second sample toan inlet region in contact with the inlet of the capillary; applying aninjection forward polarity voltage pulse to create a plug of the secondDNA fragments proximate the inlet of the capillary; and flushing thesecond sample from the inlet region.

In some implementations, applying the reverse polarity voltage pulsemoves some of the first DNA fragments that had already passed theinterrogation region to a position upstream of interrogation region.

In some implementations, the method further includes terminating thereverse polarity voltage pulse prior to introducing the second sample tothe inlet of a capillary.

In some implementations, the method further includes terminating thereverse polarity voltage pulse prior to applying the secondsubstantially constant forward polarity electrophoresis voltage to thecapillary.

In some implementations, the method further includes: (i) whileperforming electrophoresis on the first sample in the capillary,detecting an internal calibrant at the interrogation region; (ii) whileapplying the reverse polarity voltage pulse to the capillary, detectingfor a second time the internal calibrant at the interrogation region;and (iii) while applying a second substantially constant forwardpolarity electrophoresis voltage to the capillary, detecting for a thirdtime the internal calibrant at the interrogation region.

In some implementations, the method further includes interpreting theelectrophoresis of the first DNA fragments and the second DNA fragments,wherein the interpreting includes disregarding signal obtained at theinterrogation region during a period between detecting the internalcalibrant for the first time and third time.

In some implementations, the method further includes before detectingall of the second DNA fragments at the interrogation region: applying asecond reverse polarity voltage pulse to the capillary, therebytransporting at least some of the second DNA fragments in the capillarytoward the inlet of the capillary; introducing a third sample to theinlet of a capillary, the third sample including third DNA fragmentshaving a plurality of different sizes; and applying a thirdsubstantially constant forward polarity electrophoresis voltage to thecapillary to simultaneously perform electrophoresis on the third DNAfragments and the second DNA fragments.

In some implementations, applying the reverse polarity voltage pulsemoves all of the first DNA fragments that have not yet passed theinterrogation region when the reverse polarity voltage pulse was appliedto positions in the capillary such that when the second substantiallyconstant forward polarity electrophoresis voltage is applied, said firstDNA fragments that had not yet passed the interrogation region stillhave not passed the interrogation region.

In some implementations, the method further includes creating anelectropherogram of the first sample by splicing together partialelectropherograms of the first sample obtained before and after applyingthe reverse polarity voltage pulse.

In some implementations, splicing together includes removing dataobtained after a reference point in a first partial electropherogramobtained before applying the reverse polarity voltage pulse and removingdata obtained before the reference point in a second partialelectropherogram obtained after applying the reverse polarity voltagepulse. In some implementations, the method further includes identifyingthe reference point by identifying an electropherogram peak associatedwith the reference point.

Another aspect of the disclosure relates to a method of performingcapillary electrophoresis of multiple samples. The method includes:initiating electrophoresis of a first sample in a capillary containing aseparation medium, the first sample including first DNA fragments havinga plurality of different sizes; conducting steady-state transport offirst DNA fragments from the first sample by applying a substantiallyconstant forward polarity electrophoresis voltage to the capillary;before completing electrophoresis of the first sample, haltingapplication of the substantially constant forward polarityelectrophoresis voltage and thereby interrupting the steady-statetransport of the first DNA fragments in the capillary; applying areverse polarity voltage to the capillary to drive transport of thefirst DNA fragments toward an inlet of the capillary; haltingapplication of the reverse polarity voltage; introducing a second sampleto an inlet region in contact with the capillary inlet, wherein thesecond sample includes second DNA fragments having a plurality ofdifferent sizes; applying an injection forward polarity voltage pulse tocreate a plug of the second DNA fragments in the capillary, proximatethe capillary inlet; applying a second substantially constant forwardpolarity voltage to cause steady-state transport of both the first DNAfragments and the second DNA fragments in the capillary; andinterrogating the first DNA fragments and the second DNA fragments asthey reach an interrogation region proximate a distal end of thecapillary.

A further aspect of the disclosure relates to a system for performingcapillary electrophoresis of multiple samples. The system includes: (a)a capillary containing a separation medium and having inlet and distalends; (b) an inlet in communication with the capillary inlet andconfigured to receive samples containing DNA fragments having aplurality of different sizes; (c) an interrogation region configured todetect DNA fragments moving through the capillary; (d) a power sourceconfigured to apply forward and reverse polarity voltages between inletand distal ends of the capillary; and (e) logic. The control logic isconfigured for directing the following operations: introducing a firstsample to the capillary inlet, the first sample including first DNAfragments having a plurality of different sizes; performingelectrophoresis on the first sample in the capillary by applying a firstsubstantially constant forward polarity electrophoresis voltage to thecapillary while detecting some of the first DNA fragments at theinterrogation region; before all of the first DNA fragments have passedthe interrogation region, applying a reverse polarity voltage pulse tothe capillary, thereby transporting at least some of the first DNAfragments in the capillary toward the capillary inlet; thereafterintroducing a second sample to the capillary inlet, the second sampleincluding second DNA fragments having a plurality of different sizes;and applying a second substantially constant forward polarityelectrophoresis voltage to the capillary to simultaneously performelectrophoresis on the second DNA fragments and the first DNA fragments.

In some implementations, the system further includes a chassis enclosingthe capillary, the inlet region, the interrogation region, the powersupply, and the logic.

In some implementations, the system further includes a chassis enclosingthe capillary, the inlet region, the interrogation region, and the powersupply, but not the logic.

In some implementations, the system further includes logic for creatingan electropherogram of the first sample by splicing together partialelectropherograms of the first sample obtained before and after applyingthe reverse polarity voltage pulse. In some implementations, the systemfurther includes the logic is further configured to direct amplifyingthe DNA source to produce amplicons from loci of the STRs. In someimplementations, the system further includes a cartridge includingfluidic passages and a thermocycler configured to perform polymerasechain reaction.

In some implementations, the system further includes the logic isfurther configured to direct the introduction of the second sample tothe inlet of the capillary by: introducing the second sample to an inletregion in contact with the inlet of the capillary; applying an injectionforward polarity voltage pulse to create a plug of the second DNAfragments proximate the inlet of the capillary; and flushing the secondsample from the inlet region.

In some implementations, the logic is further configured to terminatethe reverse polarity voltage pulse prior to introducing the secondsample to the inlet of a capillary.

In some implementations, the logic is further configured to terminatethe reverse polarity voltage pulse prior to applying the secondsubstantially constant forward polarity electrophoresis voltage to thecapillary.

In some implementations, the logic is further configured to direct thefollowing operations: (i) while performing electrophoresis on the firstsample in the capillary, detecting an internal calibrant at theinterrogation region; (ii) while applying the reverse polarity voltagepulse to the capillary, detecting for a second time the internalcalibrant at the interrogation region; and (iii) while applying a secondsubstantially constant forward polarity electrophoresis voltage to thecapillary, detecting for a third time the internal calibrant at theinterrogation region. In some implementations, the logic is furtherconfigured to direct the following operation: interpreting theelectrophoresis of the first DNA fragments and the second DNA fragments,wherein the interpreting includes disregarding signal obtained at theinterrogation region during a period between detecting the internalcalibrant for the first time and third time.

In some implementations, the logic is further configured to direct thefollowing operation before detecting all of the second DNA fragments atthe interrogation region: applying a second reverse polarity voltagepulse to the capillary, thereby transporting at least some of the secondDNA fragments in the capillary toward the inlet of the capillary;introducing a third sample to the inlet of a capillary, the third sampleincluding third DNA fragments having a plurality of different sizes; andapplying a third substantially constant forward polarity electrophoresisvoltage to the capillary to simultaneously perform electrophoresis onthe third DNA fragments and the second DNA fragments.

In some implementations, the logic is further configured to direct thefollowing operation: creating an electropherogram of the first sample bysplicing together partial electropherograms of the first sample obtainedbefore and after applying the reverse polarity voltage pulse. In someimplementations, splicing together includes removing data obtained aftera reference point in a first partial electropherogram obtained beforeapplying the reverse polarity voltage pulse and removing data obtainedbefore the reference point in a second partial electropherogram obtainedafter applying the reverse polarity voltage pulse.

In some implementations, the logic is further configured to direct thefollowing operation: identifying the reference point by identifying anelectropherogram peak associated with the reference point.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a block diagram depicting electrophoresis apparatus suitablefor use with the processes described herein.

FIG. 1B is a schematic illustration of an analysis module useful in thesystems described herein.

FIG. 2 is a timing diagram depicting a conventional electrophoresisprocess.

FIG. 3 is a timing diagram depicting an electrophoresis process inaccordance with certain embodiments described herein.

FIG. 4A is a partial electropherogram of two samples provided in twotraces.

FIG. 4B is a partial electropherogram of three samples provided in threetraces.

TERMINOLOGY

Every embodiment of the disclosure may optionally be combined with anyone or more of the other embodiments described herein which areconsistent with that embodiment.

Whenever the term “about” or “approximately” precedes the firstnumerical value in a series of two or more numerical values or in aseries of two or more ranges of numerical values, the term “about” or“approximately” applies to each one of the numerical values in thatseries of numerical values or in that series of ranges of numericalvalues. In certain embodiments, the term “about” or “approximately”means within 10% or 5% of the specified value.

Whenever the term “at least” or “greater than” precedes the firstnumerical value in a series of two or more numerical values, the term“at least” or “greater than” applies to each one of the numerical valuesin that series of numerical values.

Whenever the term “no more than” or “less than” precedes the firstnumerical value in a series of two or more numerical values, the term“no more than” or “less than” applies to each one of the numericalvalues in that series of numerical values.

The term “sample”, as used herein, refers to a sample containingbiological material. A sample may be, e.g., a fluid sample (e.g., ablood sample) or a tissue sample (e.g., a cheek swab). A sample may be aportion of a larger sample. A sample can be a biological sample having anucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), or a protein. A sample can be a forensic sample or anenvironmental sample. A sample can be pre-processed before it isintroduced to the system; the preprocessing can include extraction froma material that would not fit into the system, quantification of theamount of cells, DNA or other biopolymers or molecules, concentration ofa sample, separation of cell types such as sperm from epithelial cells,concentration of DNA using an Aurora system (Boreal Genomics) or beadprocessing or other concentration methods or other manipulations of thesample. A sample can be carried in a carrier, such as a swab, a wipe, asponge, a scraper, a piece punched out a material, a material on which atarget analyte is splattered, a food sample, a liquid in which ananalyte is dissolved, such as water, soda. A sample can be a directbiological sample such as a liquid such as blood, semen, saliva; or asolid such a solid tissue sample, flesh or bone.

The disclosed embodiments can also be applied to process and analyze asample that has been previously preprocessed, for example, by extractionof DNA from large object such as a bed sheet or chair and otherprocessing which may include quantification of DNA concentration, cellconcentration, or other manipulations before input of the pre-processedsample into the sample cartridge.

DETAILED DESCRIPTION

A limitation on the throughput on an electrophoresis system is how oftena capillary can accept a new sample. Classically, a next sample is notinjected until all its species of interest in a first sample havemigrated past the location of detection. In, e.g., a typical STRanalysis the time until the first species arrives at the detector isabout one third the time until the slowest species arrives, hence duringa third of the time there is no information of interest collected.

One might consider using a system that interrupts the ongoing migrationof a sample and injects the next sample into the capillary such thatwhen the migration is resumed, the fastest species of the next sample donot reach the location of detection until the slowest species of thefirst sample has already arrived. In that way the time when there are nospecies of interest arriving at the detector is minimized. However, ininterrupting the migration of the first sample the important timinginformation collected at the location of detection is compromised. Inparticular the injection itself involves applying a sequence ofvoltages. Further, there is a perception that some fragments from asample might be retained in the capillary, so it is safe to fully cleara current sample before introducing a next sample.

This approach could be error prone due to various uncharacterized systemtime constants for, e.g., controlling and detecting high voltage,thermal time constants related to the joule heating in the capillaryfrom applying the high voltage, as well as any non-linearity of themigration speed with the voltage applied. As explained below, suchchallenges can be addressed by compensating for the initial high voltagepulse (often referred to as an injection pulse) and other variationsassociated with introducing the next sample.

Apparatus

In certain embodiments, the relevant components and operation of theapparatus are fairly simple and are described in connection with FIGS.1A and 2. FIG. 1A presents a block diagram of the apparatus and FIG. 2provides a conventional electropherogram timing diagram.

As shown in FIG. 1A, an electrophoresis apparatus 101 includes acapillary with gel 103 and an inlet region 105 (e.g., an inlet chamber)at the entrance of the capillary. During operation, a DNA sample isprovided to inlet region 105. The sample may be provided in a buffersolution. In the inlet region 105, the sample contacts the gel at aproximal end of the capillary. The apparatus 101 also includeselectrophoresis electrodes 107 and 109. As shown, electrode 109 isprovided with a positive charge and disposed at a distal end ofcapillary 103, while electrode 107 is provided with a negative chargeand disposed at the proximal end of the capillary. This polarity issometimes referred to as a “forward” electrophoresis voltage; theabsolute magnitudes of the potentials at the electrodes are lessrelevant than the relative difference in their potentials. A powersource 111 is configured to apply one or more defined voltage levels toone or both of electrodes 107 and 109. The power source is connected byelectrically conductive lines and/or other circuit elements to theelectrodes. Finally, electrophoresis apparatus 101 includes a detectionsystem 113 configured to detect sample species passing through aninterrogation region 115 near the distal end of capillary 103. Thedetection system detects signal emitted by species in the interrogationregion. In certain embodiments, the detection system detects fluorescentsignal emitted by species. In some implementations, the apparatusincludes components not directly connected to performingelectrophoresis. These may include a cartridge that includes fluidicpassages and a thermocycler configured to perform polymerase chainreaction. Examples of apparatus, modules, and systems suitable for usewith the present disclosure are presented in U.S. Pat. No. 8,894,946,issued Nov. 25, 2014, which is incorporated herein by reference in itsentirety.

In certain embodiments, a capillary electrophoresis system is part of ananalysis and detection module illustrated in FIG. 1B. In the depictedembodiment, a sample (e.g., amplified DNA or controls) and buffer (e.g.,electrophoresis buffer) flow through a fluidic conduit, such as a tube,from an analyte preparation module in a path that can include a denatureheater, a cathode assembly for injecting analyte into a capillary, andout to waste. A denature heater heats fluid containing DNA and denaturesstrands in double stranded DNA. The cathode assembly can include anelectrode, such as a forked electrode, connected to a source of voltage.When a sample to be analyzed is positioned for injection, the electrodecan provide voltage to inject the analyte into the capillary. Thecapillary is filled with a separation medium, such as linearpolyacrylamide (e.g., LPA V2e, available from IntegenX Inc., Pleasanton,Calif.). The capillary ends are electrically connected to a voltagesource, e.g., two electrodes, operated as an positive electrode(labelled as anode) and a negative electrode (labelled as cathode), andcapable of being powered in the reverse direction e.g., the negativeelectrode becomes the positive electrode when a power source applies apositive potential to the electrode. Separated analyte is detected witha detection module. The detection module can employ, for example, alaser and a detector, such as a CCD camera, CMOS, photomultiplier, orphotodiode. Any of these may be disposed to detect passing DNA fragmentsin an interrogation region. The anode assembly (e.g., anode cartridgeinterface) can include an anode in electrical connection with thecapillary and a source of voltage. The anode assembly also can include asource of separation medium and a source of pressure for introducingseparation medium into a capillary. The anode assembly can includeelectrophoresis buffer. The separation medium and/or the electrophoresisbuffer can be included in an anode cartridge. The anode cartridge can beconfigured for removable insertion into the anode assembly. It cancontain separation medium and/or electrophoresis buffer sufficient forone or more than one run.

In certain embodiments, the capillary electrophoresis assembly includesan injection assembly that can include a denature assembly, a cathodeassembly; a capillary assembly; an anode assembly; a capillary fillingassembly for filling a capillary with separation medium; a positioningassembly for positioning an analyte (or sample) for capillary injection;and a power source for applying a voltage between the anode and thecathode.

The capillary electrophoresis system can include one or more capillariesfor facilitating sample or product separation, which can aid inanalysis. In some embodiments, a fluid flow path directs a sample orproduct from the cartridge to an intersection between the fluid flowpath and a separation channel. (See FIG. 1B) The sample is directed fromthe fluid flow path to the separation channel, and is directed throughthe separation channel with the aid of an electric field, as can begenerated upon the application of an electrical potential across ananode and a cathode of the system (see below). U.S. Patent PublicationNo. 2011/0005932 (“UNIVERSAL SAMPLE PREPARATION SYSTEM AND USE IN ANINTEGRATED ANALYSIS SYSTEM”), which is incorporated herein by referencein its entirety, provides examples of electrophoresis capillaries foruse in analysis, as may be used with systems herein. The capillary canbe inserted into the fluidic conduit for fluidic and electriccommunication.

A cathode also can be in electric communication with the capillarythrough an electric communication with fluid in the fluidic conduit. Thecathode can be disposed in the fluidic conduit near the connection withthe capillary. For example the cathode can be positioned opposite thepoint at which the capillary connects with the fluidic conduit (e.g.,neither upstream nor downstream of the connection). This can aidinjection of the sample into the capillary and/or to provide voltage forthe electrophoresis run. In certain embodiments, the cathode cancomprise a forked electrode in which one fork is positioned upstream andone fork is positioned downstream of the point of connection of thecapillary and the fluidic conduit. In other embodiments, the cathodecomprises both a forked electrode and a third electrode positioned nearthe connection between the fluidic conduit and the capillary.

An electrophoresis sample (e.g., amplification products) can be preparedfor injection into a separation channel (e.g., a capillary) by anysuitable method. As an example, field-amplified stacking (FAS) can beperformed by positioning in an electrophoresis sample channel a dilutedmixture comprising the sample of lower salt concentration or lower ionicstrength between areas comprising an electrophoresis buffer of highersalt concentration or higher ionic strength. As another example, a bolusof a material (e.g., air) can be positioned downstream of the sample inthe sample channel, wherein the material has an electrical conductivitythat differs from the electrical conductivity of the electrophoresisbuffer or the sample, as described below. When the sample is positionedacross the separation channel, the sample can be electrokineticallyinjected into the separation channel at an appropriate voltage (e.g.,about 3 kV to about 5 kV, or about 4 kV) over an appropriate amount oftime (e.g., about 10 sec to about 20 sec, or about 15 sec).

The system may include a device for regulating the temperature of theelectrophoresis capillary or capillaries. The capillary may be disposedin an electrically insulating circuit board that has a generally curvedpath or a substantially straight path for placement of the capillary. Insome embodiments, the capillary is provided in a curvilinear path, suchas, e.g., a generally S-shaped path. The path can be distributed into aplurality of sections. Each of the sections separately regulates thetemperature in a portion of the capillary in thermal communication withthe section. Temperature may be regulated with the aid of resistiveheating, though other temperature control elements (e.g., heatingelement and/or cooling element), or devices may be used. Temperature canbe measured with the aid of a temperature sensing device, such as athermocouple, a thermistor or a resistive temperature device (RTD) ineach section. Each of the different sections includes an electrical paththat traverses the capillaries of each section. In some cases, theelectrical path traverses back and forth (e.g., in a serpentine shape inthat section). The electrical path includes one or more temperaturecontrol elements (e.g., heating elements and/or cooling elements) (e.g.,resistive heaters) for providing heat to the capillary.

A thermal sensor is in contact with each of the separately thermallyregulated areas or sections of the path. Examples of temperature sensorsare thermistors or other temperature-varying resistance, orthermocouples or other temperature-varying voltage source. In somecases, the temperature data of the separately thermally regulatedsections is not gathered by discrete temperature sensor, but by theelectrical paths themselves such as by the resistances of the electricalpaths. External temperature sensors may also be used.

Electrophoresis Process

Electrophoresis is generally conducted according a defined protocol,which may be represented via a timing diagram. See diagram 201 of FIG.2. Initially in the electrophoresis process, a sample is introduced tosystem. See sample introduction portion 203 of timing diagram 201. Thesample may be obtained from any of various sources such as crime scenesamples (e.g., bone, teeth), bodily fluid (sputum, urine, blood, semen,hair, etc.), and buccal swab samples taken from the cheek or a knownindividual. In some embodiments, the sample is preprocessed according toa standard such as described in US Patent Application Publication2016/0116439, published Apr. 28, 2016 (Kindwall et al.), which isincorporated herein by reference in its entirety. Preprocessing mayinclude operations such as lysis, DNA extraction, and DNA amplification.In some applications, preprocessing includes amplifying DNA to producethe amplicons from loci of the STRs. In some implementations, the firstDNA fragments undergoing electrophoresis are amplicons from loci of STRsin a DNA source.

In some implementations, before introduction to the inlet region (e.g.,region 105 of FIG. 1A), the sample's DNA fragment concentration isadjusted to a standard concentration and/or a standard volume. Once inthe inlet region, the sample DNA fragments in a buffer may contact anentrance to the capillary (e.g., capillary 103 of FIG. 1A), but thesample does not significantly enter the capillary gel. After the sampleis loaded into a chamber or other region at the entrance of thecapillary, a large magnitude injection voltage pulse is applied to drivethe DNA from the inlet region into the gel, where all the DNA in thesample forms a plug near the entrance. See the injection voltage portion205 of timing diagram 201.

The injection pulse is applied across two electrodes such as electrodes107 and 109 in FIG. 1A, one at the inlet of the capillary and the otherat the outlet of the capillary (distal end). As mentioned, fornegatively charged analytes such as DNA, the more positive potential isapplied to the outlet and the more negative potential is applied to theinlet.

After the injection voltage pulse ends—e.g., the voltage goes to zeromagnitude—the sample is flushed out of the inlet region and a buffer isintroduced into the region. The buffer can serve as a medium totransport of the DNA fragments through the length of the capillary. Seethe buffer introduction portion 207 of timing diagram 201.

After the buffer is introduced to the inlet region, a newelectrophoresis voltage, called the main electrophoresis voltage, isapplied and maintained for the duration of the electrophoresis process.Typically, it has a lower magnitude and longer duration than theinjection voltage pulse. See the electrophoresis voltage applicationportion 209 of timing diagram 201.

The electrophoresis voltage sets up the steady-state transport of bufferand DNA fragments through the capillary. The steady-state flow ensuresthat different alleles, which have different lengths, reach theinterrogation region near the distal end of the capillary at reasonablypredictable times.

Conventional STR electrophoresis employs many internal calibrants, whereeach calibrant is a DNA fragment of a defined size labeled with aparticular dye that is unique to the internal calibrants. Using acombination of calibrant detection and sample fragment travel time,conventional systems determine the fragment size of the allele beingread at the interrogation region.

Further details of some embodiments of an electrophoresis process arepresented in U.S. Pat. No. 8,894,946, previously incorporated herein byreference.

Staggered Electrophoresis

The disclosed system and associated method sequentially process multiplesamples in a single gel, e.g., in a single capillary. For reasonsmentioned above, putting multiple samples in a single gel was viewed asan unacceptable practice. There is a perception that some DNA fragmentscould remain stuck in the gel after a prior sample is processed, andthat the fragments remaining in the gel could interfere with the nextsample subjected to electrophoresis. However, in embodiments of thisdisclosure, this has not been observed to be problem.

1. Staggered Introduction of Samples

Two or more samples are processed serially. As indicated elsewhereherein, this disclosure refers to two sequentially processed samples, a“current” sample and a “next” sample. The samples are introduced suchthat the next sample is introduced into a gel before the current samplecompletes electrophoresis in the same gel. In other words, the nextsample is introduced to the gel before all nucleic acid fragments in thecurrent sample have passed through the capillary's interrogation region.However, the next sample is not introduced so early that its fastestmoving fragment can catch the slowest moving fragment of the currentsample. In other words, there is no overlap between the interrogation offragments of the current sample and fragments of the next sample.However, because the next sample is introduced before electrophoresis ofthe current sample is complete, the sample throughput is increasedcompared to a case in which the next sample is introduced only after theprior sample has run to completion.

This staggered introduction, and particularly the fact that the nextsample is introduced before the prior sample is run to completion,introduces certain technical challenges. Notably, the introduction ofthe next sample requires prematurely ending application of theelectrophoresis voltage for the current sample. It also requiresapplying an injection high-voltage pulse for the next sample. Both ofthese variations from the constant electrophoresis voltage affect thetransport time of the DNA fragments of the current sample and can maketiming-based interrogation of the current sample unreliable. To reducereliance on transport time, one could design a system with manyadditional internal calibrants, but these are expensive.

The potential problems of staggered electrophoresis will now beexplained in more detail. As explained, after a new sample is loadedinto the inlet region at the entrance of the capillary, a largemagnitude injection voltage pulse is applied across the electrophoresiselectrodes to inject the DNA from the sample into the gel. Through thisinjection voltage pulse, the DNA fragments of the sample form a narrowwidth plug near the entrance of the capillary. After the voltage pulseends—e.g., the voltage goes to zero or near zero magnitude—a buffer isintroduced into the region. An electrophoresis running voltage is thenapplied and maintained for the duration of the electrophoresis process.This electrophoresis running voltage sets up the steady-state transportof buffer and DNA fragments through the capillary. The steady-state flowensures that different alleles, which have different lengths, reach theinterrogation region at the distal end of the capillary at reasonablypredictable times.

However, there are nonlinearities in how the gel interacts with nucleicacid fragments of particular lengths. In particular, the travel rate isnot a linear function of fragment size. For any given gel, there arecertain well-characterized regions of fragment size where there is areasonably strong deviation from linearity. To address this challenge,conventional electrophoresis employs many internal calibrants, whereeach calibrant is a fragment of a defined size labeled with a particulardye that is unique to the internal calibrants. Using a combination ofcalibrant detection and sample fragment travel time, conventionalsystems determine the fragment size of the allele being read at theinterrogation region. For a given process design, the number of internalcalibrants is minimized to reduce cost.

Note that the number and pattern of spacings between adjacent internalcalibrants are well-defined and can be used as a ruler for determiningthe length of any allele fragments passing through the interrogationregion. To account for the above-mentioned non-linearities, the internalcalibrants are not evenly spaced.

Because electrophoresis analysis relies on steady-state flow of onesample, introduction of a second sample before the first samplecompletes can introduce errors. This is because the second samplerequires a period of no applied voltage when the second sample isloaded. It also requires an introductory voltage pulse and a secondperiod of no applied voltage when the sample is flushed. All of thiscauses the first sample to depart from steady-state flow. In theory, thefirst sample could be processed with such deviations from steady-stateflow. This would be possible by employing many additional internalcalibrants. In other words, the spacing between successive calibrantswould be consistently small enough to provide the resolution necessaryto uniquely identify all sample fragments of interest without relying ontransport time.

Unfortunately, internal calibrants are costly and using many of themsignificantly adds to the cost of sample analysis. Therefore, in variousembodiments, few, if any, additional calibrants are used. In certainembodiments, the internal calibrants span a fragment size range ofbetween about 20 base pairs and 1000 base pairs, or between about 50base pairs and 700 base pairs, or between about 100 base pairs and about500 base pairs.

To recap, certain disclosed embodiments employ a staggered introductionof successive electrophoresis samples and they do so in a manner thatdoes not use more than a defined and relatively low number or density ofinternal calibrants. In embodiments, with this constraint, the systemcompensates for the non-steady transport of fragments near theinterrogation region when the next sample is started. In some cases, thesystem accomplishes this by applying a reverse voltage pulse prior tointroduction of the next sample.

2. Reverse Polarity Voltage Pulse

Because some embodiments use only limited numbers internal calibrants,the electrophoresis runs must rely on the respective transport times ofthe DNA fragments travelling through the capillary to reach theinterrogation region. This means that the process must compensate fornon-steady-state transport of the DNA fragments through the capillary.As explained, the non-steady-state transport is caused by variations inthe electrical field applied to the capillary during the initial stagesthe next sample introduction and electrophoresis.

In various embodiments, the electrophoresis system compensates fornon-steady-state transport by stopping electrophoresis before thecurrent sample has been completely analyzed and before the next sampleis introduced, and applying a reverse polarity voltage pulse to thecapillary. In other words, a potential is applied to the electrodes suchthat the potential of the electrode at the distal end of the capillaryis relatively negative compared to the potential of the electrode at theproximal end of the capillary. This runs the DNA fragments in reversedirection, effectively backing them up in the capillary, so that whenthe next sample starts up—at which time the system produces the requiredlarge voltage variations such as the injection pulse—the DNA fragmentsalready in the capillary must move forward by some distance beforeregaining their positions prior to the reverse polarity pulse. Thus,during the initial phases of the next sample run, the DNA fragments arererun in the forward direction, retracing a portion of their migrationthrough the capillary. By the time these fragments reach theinterrogation region, they are travelling under the influence of therunning electrophoresis voltage applied for the next sample. In otherwords, they are travelling under steady-state conditions. In someembodiments, by the time the fragments reach the point at which thereverse polarity voltage pulse was applied, they are travelling underthe influence of the running electrophoresis voltage.

In one embodiment, under reverse polarity, fragments of the currentsample are moved backward a sufficient distance so that the leading edgeof the electropherogram, after the next sample injection and return torunning electrophoresis voltage, overlaps the back edge of theelectropherogram pre-stop, so that the pre- and post-stopelectropherograms can be stitched together.

The reverse pulse is applied in a manner that ensures that detection offragments from the next sample do not interfere with theelectropherogram for the current sample. In certain embodiments, thereverse pulse is timed so that the fastest fragment of the next sampledoes not catch the slowest fragment of the current sample at theinterrogation region. This may be ensured by starting the reverse pulseonly after a defined fraction of the DNA fragments from the currentsample are detected and/or running the reverse pulse for a limitedduration (and/or at a limited magnitude). In certain embodiments, acurrent sample is run to approximately 20 to 80% of completion beforethe next sample is introduced. In certain embodiments, a current sampleis run to approximately 50 to 80% of completion before the next sampleis introduced.

Considering the DNA fragments in total in the current sample, allfragments were either (a) transported and detected prior to the reversepolarity pulse using normal electrophoresis processing (i.e., they weretransported all the way through the capillary to the interrogationregion using the steady running electrophoresis voltage without beingexposed to the start-up conditions for the next sample), or (b)subjected to unsteady voltages from the next sample start up, but onlywhile they are located in positions upstream from their positions whenthey were exposed to the reverse polarity voltage pulse. When thereverse polarity pulse is large enough, the second type of fragmentsregain steady transport well before reaching the interrogation region,thereby providing some overlap in time between the pre-stop andpost-stop electrophoresis detection data. This allows the system tocreate a composite electropherogram in which distortions caused bystopping, reversing, injecting, and re-running are effectivelyeliminated, or do not significantly interfere with detection of peaksacross the composite electropherogram.

In some implementations, the resulting interrogation data is manipulatedby removing all peaks generated during a time frame starting with orjust before the initiation of the reverse polarity voltage pulse andending when the DNA fragments regain the positions they attained whenthe reverse polarity pulse was applied. In other embodiments,electropherogram data is simply not collected during the reverse pulseand/or during the initial phases of the next sample (e.g., injection).When the electropherogram data from before the reverse pulse is alignedwith electropherogram data after the injection of the next sample, theprocess is sometimes referred to as splicing two electropherogramtraces, and it will be described in more detail with reference to FIGS.4A and B.

In certain embodiments, such as depicted in FIG. 3, the sequence ofoperations (a timing diagram 301) is as follows:

a. initiate electrophoresis of a current sample in a capillary;

b. conduct steady-state transport of DNA fragments from the currentsample; this involves applying a constant electrophoresis voltage(operation 305);

c. before completing electrophoresis of the current sample, haltapplication of the electrophoresis voltage; this interrupts thesteady-state transport of the DNA fragments in the current sample(operation 307);

d. allow system to rest for a defined period, during which no voltage isapplied (operation 309);

e. apply a reverse polarity voltage to the capillary to drive transportof the DNA fragments in the opposite direction (operation 311);

f. stop application of the reverse polarity voltage (operation 313);

g. perform initiation steps for the next sample: introduce the nextsample to the inlet region, apply the initial forward voltage pulse tocreate a plug of the next sample near the inlet of the capillary, endthe initiation pulse, flush the sample from the inlet region (operation315);

h. apply steady-state running voltage to drive transport of the DNAfragments from the current sample and for the next sample in theelectrophoresis capillary (operation 317);

i. complete the interrogation of the current sample; and

j. stitch electropherogram traces to eliminate unsteady portions of theprocess.

Certain embodiments of the reverse pulse interruption electrophoresisprocess may be understood as follows. By interrupting the migration of afirst sample and then reversing the migration for some time beforeresuming migration at the original conditions, some sample fragments maypass the interrogation location multiple times: first before theinterrupt, then secondly when the reverse polarity is applied and lastlyagain when the original conditions has been regained. This process maybe conducted such that migration is occurring at steady-state conditionsduring the first and last passage, i.e., before the interruption, andafter the original condition has been regained. The electrophoresis dataup to and including the first passage is uncompromised as well as theelectrophoresis data from its last passage to any other species passinglater. By simply removing or ignoring any data between the first passageand the last, all the electropherograms obtained under steady-statetransport can reconstructed as if the interruption never happened. Insuch process, the next sample can be injected prematurely to thefinishing of the current sample as desired, without compromising thetiming information of said first sample.

In certain embodiments, the process interrupts steady-stateelectrophoresis of the current sample just after a known species thatcan be unambiguously identified has been detected in the interrogationregion. Then, the process reverses the migration to an extent such thatboth the introduction of the next sample and application of the highvoltage pulse can be performed and subsequently a steady-state isreached before the unambiguously identifiable species is detected forthe last time. One example an unambiguously identifiable specie is oneof the internal lane standards (ILS) such as the 280 bp ILS marker.

3. Characteristics of the Reverse Polarity Voltage Pulse

The reverse polarity voltage pulse begins after a fraction of thecurrent sample fragments have been detected. In some embodiments, asmentioned, it begins immediately or shortly after an easily recognizedfragment or internal calibrant is detected. This allows easy joining ofelectropherogram traces from before and after the reverse polaritypulse. In certain embodiments, the reserve pulse is initiated only afterat least about 50% of the fragments have passed the interrogationregion.

The reverse polarity voltage pulse terminates after the current sample'sundetected fragments have been sent backwards a sufficient distance thatwhen the next sample initiation is performed, all previously undetectedfragments from the current sample reach the interrogation region understeady-state conditions (those that are applied via a steady runningelectrophoresis voltage for the next sample). In other words, all DNAfragments move across the interrogation region under the influence of asteady running electrophoresis voltage, and some of them do so twice;i.e., the reverse voltage pulse builds in some lead time or distance atthe interrogation position.

In certain embodiments, the reverse polarity pulse is applied for aduration and at a magnitude sufficient to drive the current sample DNAfragments backwards by a distance such that when they are exposed to theinitiation pulse and the sample flushing portions of the next sample'sprocessing, they still have not reached the interrogation region. Whenthey finally do reach the interrogation region, they are again travelingat a steady-state, which allows them and all subsequent fragments to beread properly. In certain embodiments, at least about 5% of the entireelectropherogram (for the current sample) is backed up during thereverse pulse. In certain embodiments, at least about 10% of the entireelectropherogram (for the current sample) is backed up during thereverse pulse.

As described above, one approach to ensure that the magnitude andduration of the reverse pulse are sufficient involves detecting aparticular peak from an internal calibrant during a first pass, thendetecting a peak from the same calibrant again during application of thereverse polarity pulse, and finally detecting a third peak from the samecalibrant during application of the forward electrophoresis voltage forthe next sample.

The negative voltage pulse has a magnitude and duration sufficient toaccomplish one or more of the above objectives. If its magnitude isrelatively low, it may have to be applied for a relatively longerduration. By contrast, if its magnitude is relatively high, it may beapplied at a relatively shorter duration.

The hardware for using a reverse polarity pulse capillary can be similarto existing hardware for capillary electrophoresis. However, in certainembodiments, the hardware employs a power supply that runs in reverse orapplies a reverse bias.

Splicing Electropherogram Traces

FIGS. 4A and 4B display the output of a detector during anelectrophoresis run in which two DNA-containing samples are successivelyintroduced to the electrophoresis apparatus. The output shows multiplepeaks, each associated with a particular DNA fragment passing through aninterrogation region of the apparatus. In a color representation, thepeaks could be distinguished based on their colors, which correspond todyes affixed to individual samples or fragments. In FIG. 4A, two traces403 and 405 are shown, with each one presenting the detector outputduring constant operation of the detector. The first trace ends when thedetector is turned off (or data from the interrogation region isotherwise not collected and/or used). This is a “stop point,” whichshould be distinguished from a “splice point.” The second trace beginswhen the detector is turned on again (or data from the interrogationregion is again collected or otherwise used). In the case of sampleshaving their DNA fragments labeled with fluorescent dyes, the laser thatstimulates fluorescence of the dyes (in the interrogation region) may beturned off and then on again to end and initiate traces.

In the depicted embodiment, the first trace 403 stops before the DNAfragments of the first or current sample complete their passage throughthe capillary. Likewise the second trace 405 begins before the fragmentsof the first sample complete their passage. In between the end of thefirst trace and the start of the second trace, the voltage of theelectrophoresis apparatus changes polarity and runs in reverse. Further,between the two traces, the second sample is loaded and subjected to itsinjection voltage pulse. In the depicted example, a known DNA fragmentpeak 411 appears on both traces. This is referred to in the figure as a“reference peak.” This may serve as a splice point, as it allows thetraces to be “stitched” together to provide effectively one continuouselectropherogram. In some embodiments, peak 411 is a known to be presentin all samples; e.g., it could be a calibrant peak of a size known topass through the interrogation region at a time when, or slightly beforewhen, the next sample is to be introduced. Magnified trace segments 413show how peak 411 can be aligned to stitch together traces 403 and 405at the proper time point.

The process of depicted example may operate as follows. First the firstsample is introduced the inlet region and processed per normalelectrophoresis set up (e.g., it is subjected to an injection voltagepulse). The first sample then undergoes steady-state electrophoresis toa point until at least reference peak 411 is recorded. This producesfirst trace 403. At this point, the process prepares to receive thesecond sample. It does this by optionally turning off theelectropherogram detector (e.g., a fluorescence excitation laser isturned off) and then running electrophoresis in the reverse direction.After running in the reverse for a period sufficient to back up thefirst sample so that at least some of its fragments will again attainforward steady-state transport before they reach the interrogationregion a second time, the second sample is loaded to the inlet regionand subjected to an injection voltage pulse. At some point before, on,or shortly after applying the normal running electrophoresis voltage tothe second sample, the electrophoresis detector again begins collectingsignal (or at least the signal is made available for furtherprocessing). In the depicted example, this involves turning on thefluorescence excitation laser. This begins the process of capturingsecond trace 405. Thereafter, electrophoresis proceeds as normal, withthe first sample completing its run through the interrogation region,and the second sample beginning its run through the interrogationregion. To process the detected fragment data, and allow properinterpretation of the first sample, the two traces are aligned onreference peak 411.

FIG. 4B illustrates extension of the process to three samples. Ofcourse, the process can be extended to any number of samples so long asthe gel remains undegraded and sufficient reagent is available. FIG. 4Billustrates three separate electropherogram traces, the first one 421covering a first fraction of the first sample's fragments, the secondone 423 covering the remaining fraction of the first sample's fragmentstogether with a first fraction of the second sample's fragments, and thethird one 425 covering the remaining fraction of the second sample'sfragments together with a first fraction of the third sample'sfragments. Of course, there would be at least a fourth trace (not shown)to capture the remaining fraction of the third sample's fragments. Notethat each trace contains a reference peak 411 that allows alignment oftwo successive traces. Note also that each sample includes many primerpeaks 427 that are smaller than the allele peaks, so the primer peaksoccur earlier in the electropherogram.

Control Systems

Systems provided herein include various computational hardware andsoftware. In some embodiments, a system for sample preparation,processing and analysis (or any other system provided herein) includes acontroller with a central processing unit, memory (random-access memoryand/or read-only memory), a communications interface, a data storageunit and a display. The communications interface includes a networkinterface for enabling a system to interact with an intranet, includingother systems and subsystems, and the Internet, including the World WideWeb. The data storage unit includes one or more hard disks and/or cachefor data transfer and storage. The data storage unit may include one ormore databases, such as a relational database. In some cases, the systemfurther includes a data warehouse for storing information, such userinformation (e.g., profiles) and results. In some cases, the datawarehouse resides on a computer system remote from the system. In someembodiments, the system may include a relational database and one ormore servers, such as, for example, data servers. The system may includeone or more communication ports (COM PORTS), one or more input/output(I/O) modules, such as an I/O interface. The processor may be a centralprocessing unit (CPU) or a plurality of CPU's for parallel processing.

The system may be configured for data mining and extract, transform andload (ETL) operations, which may permit the system to load informationfrom a raw data source (or mined data) into a data warehouse. The datawarehouse may be configured for use with a business intelligence system(e.g., Microstrategy®, Business Objects®). It also can be configured foruse with a forensic database such as the National DNA Index System(NDIS)) in the USA or NDAD in the United Kingdom, State DNA IndexSystems (SDIS), or Local DNA Index Systems (LDIS) or other databasesthat contain profiles from known and unknown subjects, forensicssamples, or other sample types such as organism identifications.

Aspects of the systems and methods provided herein may be embodied inprogramming. Various aspects of the technology may be in the form ofexecutable code and/or associated data that is carried on or embodied ina type of machine readable medium. “Storage” type media may include anyor all of the tangible memory of the computers, processors or the like,or associated modules thereof, such as various semiconductor memories,tape drives, disk drives and the like, which may provide non-transitorystorage at any time for the software programming. All or portions of thesoftware may at times be communicated through the Internet or variousother telecommunication networks. Such communications, for example, mayenable loading of the software from one computer or processor intoanother, for example, from a management server or host computer into thecomputer platform of an application server. Thus, another type of mediathat may bear the software elements includes optical, electrical andelectromagnetic waves, such as used across physical interfaces betweenlocal devices, through wired and optical landline networks and overvarious air-links The physical elements that carry such waves, such aswired or wireless links, optical links or the like, also may beconsidered as media bearing the software. As used herein, unlessrestricted to non-transitory, tangible “storage” media, terms such ascomputer or machine “readable medium” refer to any medium thatparticipates in providing instructions to a processor for execution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

In some embodiments, the system includes one or more modules for sampleprocessing and/or analysis, and a controller for facilitating sampleprocessing and/or analysis. The controller can include one or moreprocessors, such as a central processing unit (CPU), multiple CPU's, ora multi-core CPU for executing machine-readable code for implementingsample processing and/or analysis. The system in some cases directs asample sequentially from one module to another, such as from a samplepreparation module to an electrophoresis module.

CONCLUSION

By maximizing the throughput of a single capillary in essence reducingthe total time to answer for the last sample, the need for additionalcapillaries to meet needs for sample to answer is reduced. For example,a single capillary system can serve a plurality of sample preparationmodules. Even when all modules have finished preparing a sample at thesame time, the time until when the last sample has been fully analyzedis still relatively short.

Systems and methods provided herein, including the components of suchsystems and various routines of such methods, may be combined with ormodified by other systems and methods. In some situations, theabove-described systems may be combined or modified by the systemsdescribed in U.S. Patent Publication No. 2011/0005932 to Jovanovich etal. (“UNIVERSAL SAMPLE PREPARATION SYSTEM AND USE IN AN INTEGRATEDANALYSIS SYSTEM”) (“Jovanovich”), which is entirely incorporated hereinby reference in its entirety.

It should be understood from the foregoing that, while particularimplementations have been illustrated and described, variousmodifications may be made thereto and are contemplated herein. It isalso not intended that the invention be limited by the specific examplesprovided within the specification. While the invention has beendescribed with reference to the aforementioned specification, thedescriptions and illustrations of embodiments of the invention hereinare not meant to be construed in a limiting sense. Furthermore, it shallbe understood that all aspects of the invention are not limited to thespecific depictions, configurations or relative proportions set forthherein which depend upon a variety of conditions and variables. Variousmodifications in form and detail of the embodiments of the inventionwill be apparent to a person skilled in the art. It is thereforecontemplated that the invention shall also cover any such modifications,variations and equivalents.

1. A method of performing capillary electrophoresis of multiple samples, the method comprising: introducing a first sample to an inlet of a capillary containing a separation medium, the first sample comprising first DNA fragments having a plurality of different sizes; performing electrophoresis on the first sample in the capillary, wherein the electrophoresis comprises applying a first substantially constant forward polarity electrophoresis voltage to the capillary while detecting some of the first DNA fragments at an interrogation region proximate a distal end of the capillary; before all of the first DNA fragments have passed the interrogation region, applying a reverse polarity voltage pulse to the capillary, thereby transporting at least some of the first DNA fragments in the capillary toward the inlet of the capillary, and thereafter introducing a second sample to the inlet of a capillary, the second sample comprising second DNA fragments having a plurality of different sizes; and applying a second substantially constant forward polarity electrophoresis voltage to the capillary to simultaneously perform electrophoresis on the second DNA fragments and the first DNA fragments.
 2. The method of claim 1, further comprising introducing a plurality of internal calibrants, along with the first sample, to the inlet of the capillary, wherein each internal calibrant comprises a labeled DNA fragment of distinct size.
 3. The method of claim 1, wherein the first DNA fragments comprise amplicons from loci of STRs in a DNA source. 4.-6. (canceled)
 7. The method of claim 1, wherein introducing the second sample to the inlet of the capillary comprises: introducing the second sample to an inlet region in contact with the inlet of the capillary; applying an injection forward polarity voltage pulse to create a plug of the second DNA fragments proximate the inlet of the capillary; and flushing the second sample from the inlet region.
 8. The method of claim 1, wherein applying the reverse polarity voltage pulse moves some of the first DNA fragments that had already passed the interrogation region to a position upstream of interrogation region.
 9. The method of claim 1, further comprising terminating the reverse polarity voltage pulse prior to introducing the second sample to the inlet of a capillary.
 10. The method of claim 1, further comprising terminating the reverse polarity voltage pulse prior to applying the second substantially constant forward polarity electrophoresis voltage to the capillary.
 11. The method of claim 1, further comprising: (i) while performing electrophoresis on the first sample in the capillary, detecting an internal calibrant at the interrogation region; (ii) while applying the reverse polarity voltage pulse to the capillary, detecting for a second time the internal calibrant at the interrogation region; and (iii) while applying a second substantially constant forward polarity electrophoresis voltage to the capillary, detecting for a third time the internal calibrant at the interrogation region.
 12. The method of claim 11, further comprising interpreting the electrophoresis of the first DNA fragments and the second DNA fragments, wherein the interpreting comprises disregarding signal obtained at the interrogation region during a period between detecting the internal calibrant for the first time and third time.
 13. The method of claim 1, further comprising before detecting all of the second DNA fragments at the interrogation region: applying a second reverse polarity voltage pulse to the capillary, thereby transporting at least some of the second DNA fragments in the capillary toward the inlet of the capillary; introducing a third sample to the inlet of a capillary, the third sample comprising third DNA fragments having a plurality of different sizes; and applying a third substantially constant forward polarity electrophoresis voltage to the capillary to simultaneously perform electrophoresis on the third DNA fragments and the second DNA fragments.
 14. The method of claim 1 wherein applying the reverse polarity voltage pulse moves all of the first DNA fragments that have not yet passed the interrogation region when the reverse polarity voltage pulse was applied to positions in the capillary such that when the second substantially constant forward polarity electrophoresis voltage is applied, said first DNA fragments that had not yet passed the interrogation region still have not passed the interrogation region.
 15. The method of claim 1, further comprising creating an electropherogram of the first sample by splicing together partial electropherograms of the first sample obtained before and after applying the reverse polarity voltage pulse.
 16. The method of claim 15, wherein splicing together comprises removing data obtained after a reference point in a first partial electropherogram obtained before applying the reverse polarity voltage pulse and removing data obtained before the reference point in a second partial electropherogram obtained after applying the reverse polarity voltage pulse.
 17. (canceled)
 18. A method of performing capillary electrophoresis of multiple samples, the method comprising: initiating electrophoresis of a first sample in a capillary containing a separation medium, the first sample comprising first DNA fragments having a plurality of different sizes; conducting steady-state transport of first DNA fragments from the first sample by applying a substantially constant forward polarity electrophoresis voltage to the capillary; before completing electrophoresis of the first sample, halting application of the substantially constant forward polarity electrophoresis voltage and thereby interrupting the steady-state transport of the first DNA fragments in the capillary; applying a reverse polarity voltage to the capillary to drive transport of the first DNA fragments toward an inlet of the capillary; halting application of the reverse polarity voltage; introducing a second sample to an inlet region in contact with the capillary inlet, wherein the second sample comprises second DNA fragments having a plurality of different sizes; applying an injection forward polarity voltage pulse to create a plug of the second DNA fragments in the capillary, proximate the capillary inlet; applying a second substantially constant forward polarity voltage to cause steady-state transport of both the first DNA fragments and the second DNA fragments in the capillary; and interrogating the first DNA fragments and the second DNA fragments as they reach an interrogation region proximate a distal end of the capillary.
 19. A system for performing capillary electrophoresis of multiple samples, the system comprising: (a) a capillary containing a separation medium and having inlet and distal ends; (b) an inlet in communication with the capillary inlet and configured to receive samples containing DNA fragments having a plurality of different sizes; (c) an interrogation region configured to detect DNA fragments moving through the capillary; (d) a power source configured to apply forward and reverse polarity voltages between inlet and distal ends of the capillary; and (e) logic for directing the following operations: introducing a first sample to the capillary inlet, the first sample comprising first DNA fragments having a plurality of different sizes; performing electrophoresis on the first sample in the capillary by applying a first substantially constant forward polarity electrophoresis voltage to the capillary while detecting some of the first DNA fragments at the interrogation region; before all of the first DNA fragments have passed the interrogation region, applying a reverse polarity voltage pulse to the capillary, thereby transporting at least some of the first DNA fragments in the capillary toward the capillary inlet, and thereafter introducing a second sample to the capillary inlet, the second sample comprising second DNA fragments having a plurality of different sizes; and applying a second substantially constant forward polarity electrophoresis voltage to the capillary to simultaneously perform electrophoresis on the second DNA fragments and the first DNA fragments. 20.-21. (canceled)
 22. The system claim 19, further comprising logic for creating an electropherogram of the first sample by splicing together partial electropherograms of the first sample obtained before and after applying the reverse polarity voltage pulse.
 23. The system claim 19, wherein the logic is further configured to direct amplifying the DNA source to produce amplicons from loci of the STRs, the system further comprising a cartridge comprising fluidic passages and a thermocycler configured to perform polymerase chain reaction. 24.-27. (canceled)
 28. The system of claim 19, wherein the logic is further configured to direct the following operations: (i) while performing electrophoresis on the first sample in the capillary, detecting an internal calibrant at the interrogation region; (ii) while applying the reverse polarity voltage pulse to the capillary, detecting for a second time the internal calibrant at the interrogation region; and (iii) while applying a second substantially constant forward polarity electrophoresis voltage to the capillary, detecting for a third time the internal calibrant at the interrogation region, wherein the logic is further configured to direct the following operation: interpreting the electrophoresis of the first DNA fragments and the second DNA fragments, wherein the interpreting comprises disregarding signal obtained at the interrogation region during a period between detecting the internal calibrant for the first time and third time. 29.-30. (canceled)
 31. The system of claim 19, wherein the logic is further configured to direct the following operation: creating an electropherogram of the first sample by splicing together partial electropherograms of the first sample obtained before and after applying the reverse polarity voltage pulse.
 32. The system of claim 31, wherein splicing together comprises removing data obtained after a reference point in a first partial electropherogram obtained before applying the reverse polarity voltage pulse and removing data obtained before the reference point in a second partial electropherogram obtained after applying the reverse polarity voltage pulse.
 33. (canceled) 